General Background. In the past decade it has become apparent that many diseases result from genetic alterations in signaling pathways. These include diseases related to unregulated cell proliferation such as cancers, atherosclerosis and psoriasis as well as inflammatory conditions such as sepsis, rheumatoid arthritis and tissue rejection. The finding that these proliferative diseases are based on genetic defects refocused the medical community to seek new modalities for disease management which essentially consist of designing drugs which modulate cell signaling. In order to develop highly specific drugs, i.e., drugs which potently interfere with uncontrolled cell proliferation but have low toxicity or side effects, it is crucial to identify the genes encoding polypeptides involved in the cellular signal transduction pathways whose aberrant function may result in the loss of growth control.
Although tremendous progress in understanding relevant signal transduction pathways has been made in recent years, it is quite clear that many of the genes involved in the development of proliferative disorders, referred to herein generally as "cell proliferation genes", remain to be discovered.
Cell Proliferation Genes. Genes whose aberrant expression or function may contribute to cell proliferation disorders fall into two general categories: (1) dominant transforming genes, including oncogenes, and (2) recessive cell proliferation genes, including tumor suppressor genes and genes encoding products involved in programmed cell death ("apoptosis").
Oncogenes generally encode proteins that are associated with the promotion of cell growth. Because cell division is a crucial part of normal tissue development and continues to play an important role in tissue regeneration, oncogene activity, properly regulated, is essential for the survival of the organism. However, inappropriate expression or improperly controlled activation of oncogenes may drive uncontrolled cell proliferation and result in the development of severe diseases, such as cancer. Weinberg, 1994, CA Cancer J. Clin. 44:160-170.
Tumor suppressor genes, on the other hand, normally act as "brakes" on cell proliferation, thus opposing the activity of oncogenes. Accordingly, inactivation of tumor suppressor genes, e.g., through mutations or the removal of their growth inhibitory effects may result in the loss of growth control, and cell proliferative diseases such as cancer may develop. Weinberg, 1994, CA Cancer J. Clin. 44:160-170.
Related to tumor suppressor genes are genes whose product is involved in the control of apoptosis; rather than regulating proliferation of cells, they influence the survival of cells in the body. In normal cells, surveillance systems are believed to ensure that the growth regulatory mechanisms are intact; if abnormalities are detected, the surveillance system switches on a suicide program that culminates in apoptosis.
Several genes that are involved in the process of apoptosis have been described. See, for example, Collins and Lopez Rivas, 1993, TIBS 18:307-308; Martin et al., 1994, TIBS 19:26-30. Gene products which have been implicated in the control of or participation in apoptosis include bcl-2 (Korsymeyer, 1992, Immunol. Today 13:285-288), c-myc (Shi et al., 1992, Science 257:212-214; Evan et al., 1992, Cell 69:119-128), p53 (Rotter et al., 1993, Trends Cell. Biol. 3:46-49), TRPM-2/SGP (Kryprianou et al., 1991, Cancer Res. 51:162-166), and Fas/APO-1 (Itoh et al., 1991, Cell 66:233-243). Cells that are resistant to apoptosis have an advantage over normal cells, and tend to outgrow their normal counterparts and dominate the tissue. As a consequence, inactivation of genes involved in apoptosis may result in the progression of tumors, and, in fact, is an important step in tumorigenesis.
Mutations in tumor suppressor genes and genes encoding products involved in the control of apoptosis are typically recessive; i.e., both copies of the gene, the maternally inherited copy and the paternally inherited one, must be inactivated by mutation to remove the effect of the gene product. Usually, a single functional copy of such genes is sufficient to maintain tumor suppression. Predisposition to certain hereditary cancers involves mutant tumor suppressor genes. For example, if an individual inherits a single defective tumor suppressor gene from her father, initially her health will be uncompromised, since each cell still contains a functional copy of the gene inherited from her mother. However, as cells divide, mutations accumulate. Thus, at one point, the remaining normal copy in a cell may be inactivated by mutation to remove the function of the tumor suppressor, thereby completing one of the steps toward tumor formation. Such a cell may give rise to descendant cells which represent the early stages of cancer.
Of course, individuals who inherit a full normal complement of tumor suppressor genes can develop cancer as well. However, because two inactivating mutations are required, the development of the disease is much less frequent in such "normal" individuals, i.e., not predisposed to cancer.
Tumor suppressor genes and oncogenes participate in growth control pathways in normal cells in such a way that the appropriate level of cell division is maintained. Disruption of these pathways by mutation of the component genes, oncogenes or tumor suppressor genes, is the underlying cause of cancer. Growth control in complex organisms like humans is a very important and complicated process. Thus, multiple genetic pathways for growth control are involved. Some pathways operate in all cell types in the body. Other pathways are much more specific and function only in certain cells.
Discovery Of Cell Proliferation Genes. Oncogenes and tumor suppressor genes have traditionally been identified by different methods. However, each of the approaches currently employed for the identification and isolation of cell proliferation genes has limitations on the types of genes that can be retrieved.
A first approach involves the detection and identification of transforming retroviruses and chromosomal translocations in tumors, which has provided the means to identify dozens of oncogenes. Bishop, 1983, Annu. Rev. Biochem. 52:350-354; Stehelin et al., 1976, Nature 260:170-173; Bishop, 1987, Science 235:305-311. However, this strategy is largely limited to the identification of dominant oncogenes and it rarely leads to the identification of tumor suppressor genes since inappropriate tumor suppressor functions are recessive. Moreover, viral spread is not facilitated by decreased cell growth, thus it serves little purpose for viruses to transduce tumor suppressor genes. Similarly, viral insertion or chromosomal translocations are single events. Thus, dominant changes are far more likely to be manifested than recessive changes.
A second traditional method for identifying cell proliferation genes has been the genetic analysis of kindreds, followed by positional cloning. Kindred analysis is, in principle, suited both for the identification of oncogenes as well as recessive cell proliferation genes, including tumor suppressor genes and/or genes encoding products involved in the control of apoptosis. Through kindred analysis many recessive cell proliferation genes have been uncovered, including APC (Nishisho et al., 1991, Science 253:665-669), NFI (Xu et al., 1990, Cell 62:599-608), NF2 (Rouleau et al., 1993, Nature 363:515-521), RB (Friend et al., 1986, Nature 343:643-646), MLM (Cannon-Albright et al., 1992, Science 258:1148-1152; Kamb et al., 1994, Science 264:436-440), BRCA1 (Hall et al., 1990, Science 250:1684-1989; Miki et al., 1994, Science 266:66-71), BRCA2 (Wooster et al., 1994, Science 265:2088-2090; Wooster et al., 1995, Nature 378:789-792; Tavtigian et al., 1996, Nature Genetics 12:1-6), WT1 (Francke et al., 1979, Cytogenet. Cell Genet. 24:185-192; Gessler et al., 1990, Nature 343:774-778), and VHL (Latif et al., 1993, Science 260:1317-1320). However, a major disadvantage of the analysis of kindreds is that it is rather slow and limited, because the identification of cell proliferation genes depends on the existence of chance mutations that become established in the cell population, and cause an increased risk that is dramatic enough to be visible above the level of nonhereditary (sporadic) cancer in the population. Kruglyak et al., 1995, Am. J. Hum. Genet. 57:439-454; Kruglyak et al., 1995, Am. J. Hum. Genet. 56:1212-1223.
A third approach traditionally pursued to identify and isolate cell proliferation genes is the analysis of homozyous or hemizygous genetic lesions in tumor cells. These lesions include regions of loss of heterozygosity (LOH) or homozygous deletions. Horuk et al., 1993, J. Biol. Chem. 268:541-546.
Finally, a method which has been employed for isolating growth control genes of the tumor suppressor class involves the selection of variants that have lost certain malignancy traits, namely "revertants". Such revertant lines, however, are typically difficult to identify and separate from the majority of rapidly growing parental cells. Still, a number of such revertants have been isolated from populations of cells transformed by a variety of oncogenes and subsequent treatment with various cytotoxic agents which are toxic to growing cells or cancer cells. Fischinger et al., 1972, Science 176:1033-1035; Greenberger et al., 1974, Virology 57:336-346; Ozanne et al., 1974, J. Virol. 14:239-248; Vogel et al., 1974, J. Virol. 14:1404-1410; Cho et al., 1976, Science 194:951-953; Steinberg et al., 1978, Cell 13:19-32; Maruyama et al., 1981, J. Virol. 37:1028-1043; Varmus et al., 1981, Cell 25:23-26; Varmus et al., 1981, Virology 108:28-46; Mathey-Prevot et al., 1984, J. Virol. 50:325-334; Wilson et al., 1986, Cell 44:477-487; Stephenson et al., 1973, J. Virol. 11:218-222; Sacks et al., 1979, Virology 97:231-240; Inoue et al., 1983, Virology 125:242-245; Norton et al., 1984, J. Virol. 50:439-444; Ryan et al., 1985, Mol. Cell. Biol. 5:3477-3582. Usually, cells are exposed to these agents under such conditions where cells that have reacquired a non-transformed phenotype are contact inhibited, and hence, are less susceptible to these cytotoxic agents, leading to preferential elimination of the transformed parental cells and, after several cycles, the isolation of morphologic revertants.
In addition to being both inefficient and time consuming, the above described selection for tumor suppressor genes is based on differential growth parameters of normal versus transformed cells and hence may preclude the isolation of certain classes of revertants. Moreover, the selection procedure itself may induce epigenetic changes or changes in the number of chromosomes. Furthermore, if the cytotoxic agents used are themselves mutagenic, then their continuous presence during the selection period may generate a revertant phenotype resulting from multiple mutational events. While any of these mechanisms may result in the production of a revertant phenotype, the nature of these genetic or epigenetic changes may preclude their analysis by gene transfer experiments.
Obviously, the most constraining factor for the utility of tumor cells in gene discovery is the lack of powerful selection procedures allowing the identification of new genes. It is well recognized that there is a need for a rapid and efficient selection procedure that would permit the isolation of tumor cell revertants resulting from a single mutational event. With this objective, Zarbl et al. developed an alternative assay for the selection of revertant tumor cells. Zarbl et al., 1991, Environmental Health Perspectives 93:83-89. This selection protocol is based on the prolonged retention of a fluorescent molecule within the mitochondria of a number of transformed cells relative to non-transformed cells. Indeed, in a significant number of cases, retention of fluorescent molecules within mitochondria seems coupled to transformation. However, because the prolonged dye retention phenotype is neither essential nor sufficient for cell transformation, this approach is limited to some specific types of mechanisms of transformation.
Other methods which have been used to search for cell proliferation genes involve biochemical approaches underlying analysis of cell cycle regulators (Serrano et al., 1993, Nature 366:704-707; Xiong et al., 1993, Nature 366:701-704), random sequencing of expressed sequence tags (ESTs) and homology comparisons (Lennon et al., 1996, Genomics 33:151-152), and methods for identifying differentially expressed genes, such as differential display (Liang et al., 1995, Methods Enzymol. 254:304-321). None of these approaches, however, offers a way to directly assess the function of the genes. Instead, candidates are identified based on a presumed (or identifiable) biochemical function or on an abnormal pattern of expression. These candidates are then tested further for involvement in cancer. Such tests include either mutational alteration in primary cancers or cell lines, experiments using somatic cells (for example, to determine the effect of ectopic expression), or experiments in transgenic mice or knockout mice containing inactivated genes.
It is apparent that these selection methods have a number of drawbacks and limitations. Therefore it is desirable, and the objective of the present invention, to develop rapid and efficient selection procedures that would permit the identification and isolation of large numbers of novel genes, particularly cell proliferation genes, based on functional analysis. In accordance with its objective, the present invention provides efficient selection systems which permit the isolation of growth-proficient revertants resulting from a single mutational event in growth arrested cells.